Rapid serological test for paratuberculosis using fluorescence polarization technology

ABSTRACT

The present invention provides an assay for detection of serum antibodies to  M. paratuberculosis.  A tracer, comprising a carbohydrate antigen isolated from  M. paratuberculosis  that is conjugated to a fluorophore, is added to a serum sample from an animal to form a mixture. The fluorescence polarization of the mixture is then measured. The presence of serum antibodies to  M. paratuberculosis  is indicated by a fluorescence polarization value of the mixture that is higher than the fluorescence polarization value of a control. The present invention further provides a tracer for use in a fluorescence polarization assay for antibodies specific for  M. paratuberculosis.  The tracer comprises a carbohydrate antigen isolated from  M. paratuberculosis  and conjugated to a fluorophore, such that the tracer is able to bind to antibodies specific for  M. paratuberculosis  to produce a detectable change in fluorescence polarization.

CROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority from U.S. Patent App. No.60/335,253, filed 31 Oct. 2001. All patents, patent applications, aswell as all other scientific and/or technical writings referred toherein are incorporated by reference to the extent that they are notcontradictory.

BACKGROUND OF HE INVENTION

[0002] 1. Field of the Invention

[0003] This invention relates to the field of diagnostic assays. Moreparticularly, this invention relates to a homogeneous assay that usesfluorescence polarization technology for the detection of antibodies toMycobacterium paratuberculosis in bovine sera.

[0004] 2. Description of Related Art

[0005] Currently, ELISA methods are used to detect Mycobacteriumparatuberculosis infection in animals, such as cattle. However, thereare a number of disadvantages associated with ELISA techniques. Forexample, ELISA methods are undesirably labor intensive, in that theytypically involve several washings, liquid transfers, and incubationtimes.

[0006] Accordingly, there is a need in the art for a serological testfor M. paratuberculosis that is rapid and easy to perform.

SUMMARY OF THE INVENTION

[0007] In a first principal aspect, the present invention provides anassay for detection of serum antibodies to M. paratuberculosis. Atracer, comprising a fluorophore conjugated to an antigen isolated fromM. paratuberculosis, is added to a serum sample from an animal to form amixture. The fluorescence polarization of the mixture is then measured.The presence of serum antibodies to M. paratuberculosis is determinedfrom the measured fluorescence polarization of the mixture, e.g., thatit is higher than that of a control. The tracer can be a carbohydrateantigen isolated from M. paratuberculosis.

[0008] In a second principal aspect, the present invention provides atracer for use in a fluorescence polarization assay for antibodiesspecific for M. paratuberculosis. The tracer can comprise a carbohydrateantigen isolated from M. paratuberculosis. The tracer is conjugated to afluorophore, such that the tracer is able to bind to antibodies specificfor M. paratuberculosis to produce a detectable change in fluorescencepolarization.

DETAILED DESCRIPTION OF THE INVENTION

[0009] This assay utilizes an antigen that has been purified from M.paratuberculosis and subsequently labeled with a fluorophore. Theantigen can be a carbohydrate antigen. To perform this assay, serum isdiluted in phosphate buffered saline (PBS) supplemented with 0.1% sodiumazide and 0.05% lithium dodecyl sulfate (PBSALDS), and a baselinereading is obtained in a fluorescence polarization analyzer (FPM-1,Diachemix Corporation). The fluorescently-labeled antigen is then addedto the test tube containing the diluted serum and the fluorescencepolarization reading is obtained. The presence of antibody specific toM. paratuberculosis is indicated by a fluorescence polarization valuethat is higher than that obtained with a diluent buffer or a knownnegative serum control.

[0010] Compared to the ELISA, this assay is rapid, and technicallysimple to perform since it requires the addition of only a singlereagent, and does not involve any separation or washing steps.Typically, results are obtained in minutes.

EXAMPLE 1 Culture Conditions

[0011]Mycobacterium paratuberculosis, Strains II, II, IV, V, C286 andC300 (culture collection of Animal Diseases Research Institute, Nepean,Ontario, Canada) were each grown in Reids medium and in Longs medium for7 to 10 days at 37° C. The seed culture grown in Longs medium was thenused to inoculate multiple 1 liter flasks containing 500 mL of Longsmedium and the seed culture grown on Reids medium was used to inoculatemultiple 1 liter flasks containing 500 mL of Reids medium. The flaskswere then incubated at 37° C. for 90 days.

EXAMPLE 2 Preparation of Labeled Antigen

[0012] The 90-day cultures of all of the strains of M. paratuberculosisdescribed, grown in both media, were pooled in a glass bottle and themixture was adjusted to pH 8 using 4 N sodium hydroxide. This mixturewas stored at 4° C. for 2 weeks with shaking every 2 to 3 days. Themixture was then autoclaved for 3 hours in flowing steam after which thebottle was left undisturbed for 2 days to allow the cells to settle tothe bottom. The fluid was then siphoned, after which the cells werefiltered through sterile Whatman #2 paper. The cells were then dried,weighed, and stored at −20° C. Aliquots of cells were thawed overnightat 4° C. and water was added to make a slurry. Liquid phenol (90%) wasthen added to the slurry to yield a final phenol concentration of 30.3%(vol/vol). The slurry was next homogenized with a Polytron Homogenizerfitted with a PT 20 probe (Bricann Instruments, Ontario, Canada) for 2minutes at room temperature. The homogenate was stirred at roomtemperature for 30 minutes and then centrifuged (30,000×g, for 30minutes at 4° C.) using a swinging bucket rotor. After centrifugation,most of the aqueous phase from each tube was aspirated with a Pasteurpipette and pooled. Water (5 ml) was then added to each tube and mixedgently with the remaining aqueous phase. The tubes were re-centrifugedas before, the aqueous phase removed and pooled with the first aqueousphase extract. The pool of the aqueous phase extracts was then dialyzed(3000 kdalton molecular weight cut off) against tap water for 24 hoursand then against distilled water for a further 48 hours. The extract wasthen concentrated approximately 15-fold with an Amicon UltrafiltrationCell (Millipore, Ottawa, Canada) fitted with an Amicon YM-1 membrane(Millipore). An aliquot (600 μl) of this extract was mixed with 1Msodium hydroxide (60 μl) and incubated at 37° C. for 1 hour. The mixturewas then added to 2 ml of Polymixin B agarose (Sigma-Aldrich CanadaLimited, Oakville, Ontario, Canada), which was washed prior to use withphosphate buffered saline (PBS, 0.01M sodium phosphate +0.85% sodiumchloride, pH 7.2). The Polymixin B agarose-antigen mixture was incubatedat 37° C. for 2 hours. The mixture was then centrifuged (15,000×g, for 2minutes) and the supernatant was removed. The supernatant (approximately600 μl) was added to 1.2 mg of fluorescein isothiocyanate Isomer I(FITC, Sigma) and incubated at 37° C. for 1 hour. The labeled antigenwas then added to a Sephadex G-25 Pine (Pharmacia, Baie D'Urfe, Quebec,Canada) column (1×25 cm) pre-equilibrated with 0.1 M sodium phosphatebuffer pH 7.0. The FITC-labeled antigen was then separated from the freeFITC by elution with the 0.1M phosphate buffer. The fractions weremonitored at a wavelength of 492 nm. Two peaks were obtained andfractions in the first peak were pooled and used as the labeled antigenin the fluorescence polarization assay for detection of antibodiesspecific to M. paratuberculosis.

EXAMPLE 3 Procedure for Performing the Fluorescence Polarization Assay

[0013] Bovine serum was diluted 1:25 in PBS supplemented with 0.1%sodium azide (Sigma) and 0.05% lithium dodecyl sulfate (Sigma)(PBSALDS). A baseline reading was then obtained in a FluorescencePolarization Analyzer (FPM-1, Diachemix Corporation, Grayslake, Ill.).An aliquot of FITC-labeled antigen (which was pre-determined to yield atotal intensity value of approximately 300,000) was then added to thediluted serum. The fluorescence polarization reading was then obtained.It was found that the presence of specific antibody gave a fluorescencepolarization value higher than that obtained with a diluent buffer or aknown negative serum control.

EXAMPLE 4 Detection of M. paratuberculosis Antibodies in InfectedAnimals

[0014] Sera from over a thousand M. paratuberculosis-infected andnon-infected cattle were obtained by procedures known in the art Thesera were tested for M. paratuberculosis antibodies using the tracerprepared as described above in the fluorescence polarization assaydescribed above. The sera were also tested for M. paratuberculosisantibodies using an ELISA assay.

[0015] The ELISA method detected 23 M. paratuberculosis-infected sera;the fluorescence polarization method detected 20 of those 23 M.paratuberculosis-infected sera. Thus, the relative sensitivity of thefluorescence polarization method was about 87%.

[0016] 1013 sera tested negative using the ELISA method; 1003 of those1013 non-infected sera also tested negative using the fluorescencepolarization method. Thus, the relative specificity of the fluorescencepolarization method was about 99%

[0017] The foregoing description of the invention is presented forpurposes of illustration and description, and is not intended, norshould be construed, to be exhaustive or to limit the invention to theprecise forms disclosed. The description was selected to best explainthe principles of the invention and practical application of theseprinciples to enable others skilled in the art to best utilize theinvention in various embodiments and with various modifications as aresuited to the particular use contemplated. It is intended that the scopeof the invention not be limited by the specification, but defined by theclaims.

We claim:
 1. A method for detecting antibodies to M. paratuberculosis,the method comprising: adding a tracer to a sample from an animal toform a mixture, wherein the tracer comprises a fluorophore conjugated toan antigen isolated from M. paratuberculosis; measuring the fluorescencepolarization of the mixture; and detecting the presence of antibodies toM. paratuberculosis in the mixture from the measured fluorescencepolarization of the mixture.
 2. The method of claim 1, furthercomprising: measuring the fluorescence polarization of a control; andcomparing the fluorescence polarization of the mixture with thefluorescence polarization of the control; and
 3. The method of claim 1,wherein the sample is serum.
 4. The method of claim 1, wherein thefluorophore is fluorescein isothiocyanate.
 5. A tracer for detectingantibodies to M. paratuberculosis in a fluorescence polarization assay,the tracer comprising: a fluorophore conjugated to an antigen isolatedfrom M. paratuberculosis by: (a) growing cultures of M.paratuberculosis; (b) recovering M. paratuberculosis cells from theculture; (c) adding liquid phenol to the cells to form a mixture; (d)homogenizing the mixture; (e) isolating the aqueous phase from thehomogenized mixture; (f) dialyzing the aqueous phase; (g) concentratingthe aqueous phase; (h) adding sodium hydroxide to the aqueous phase; (i)adding polymyxin B agarose to the aqueous phase; (j) removing thepolymyxin B agarose from the aqueous phase; and (k) recovering theaqueous phase containing the antigen.
 6. The tracer of claim 5, whereinthe tracer comprises a fluorophore conjugated to a carbohydrate antigenisolated from M. paratuberculosis.
 7. The tracer of claim 6, wherein thetracer binds to antibodies specific for M. paratuberculosis to produce adetectable change in fluorescence polarization.
 8. The tracer of claim7, wherein the fluorophore is fluorescein isothiocyanate.
 9. A kit fordetecting M. paratuberculosis antibodies, wherein the kit comprises atracer as in claim 5 and wherein the antibodies are detected byfluorescence polarization.